Highly Sensitive and Robust Linear Probe for Detection of mRNA in Cells

A stemless linear probe was designed that robustly detects mRNA in cells with high sensitivity. The probe is modified at some positions with base surrogates prepared from D-threoninol, with anthraquinone moieties near the 5'- and 3'-termini, and with perylene moieties. Even in cell lysate that involves various proteins and enzymes, background emission was very low. When the probe was hybridized with RNA, chromophores are intercalated between the base pairs, resulting in a remarkable light-up signal. The signal-to-background ratio was as high as 1600 under our standard buffer conditions. In the HeLa cell lysate, the linear probe had sufficient signal-to-background ratio (S/B= 40) for reliable mRNA detection. No degradation was observed after a 24 h incubation in HeLa cell lysate. In cells, a probe designed to target DsRed resulted in distinct blue fluorescence only in cells transfected with plasmid encoding DsRed; no fluorescence was observed in control cells.